UNIT 10.8 Immunoblotting and Immunodetection
Published Online: 1 MAY 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Gallagher, S., Winston, S. E., Fuller, S. A. and Hurrell, J. G. 2004. Immunoblotting and Immunodetection. Current Protocols in Molecular Biology. 66:III:10.8:10.8.1–10.8.24.
- Published Online: 1 MAY 2004
- Published Print: APR 2004
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Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.
- western blot;
- horseradish peroxidase;
- alkaline phosphatase;